Phakopsora pachyrhizi fungi causes Asian soybean rust, limiting soybean production and resulting in losses of up to 80% if no control strategies are applied. By using a transcriptome datasets, we investigated the regulation of genes of the mevalonate pathway under different biotic stresses. We studied the impact of P. pachyrhizi infection in vivo expression profile of genes involved in terpenoid and glyceollin biosynthesis in genotypes harboring different resistance genes (Rpp), and across the infection cycle. In addition, we used UPLC and UPGC analysis to evaluate glyceollin and VOC production, respectively, to identify metabolites associated with soybean responses to pathogen infection. The regulation of soybean genes involved in terpene production was influenced by genotypes, depending on the Rpp gene, while glyceollin was induced in all genotypes. Furthermore, a sesquiterpene was identified as a potential marker associated with rust symptoms on soybean.Terpenes produced by plants comprise a diverse range of secondary metabolites, including volatile organic compounds (VOCs). Terpene VOC production may be altered after damage or by biological stimuli such as bacterial, fungal and insects, and subsequent triggering of plant defense responses. These VOCs originate in plants from two independent pathways: the mevalonate and the methylerythritol phosphate pathways, which utilize dimethylallyl and isopentenyl diphosphates to form the terpenoidal precursors.  These compounds constitute some signaling molecules in plants and are commonly produced in response to bacteria, fungi and insects infections  formed from the combination of two or more isoprenyl units, with the molecular formula  pathway, located in the plastid, and the mevalonate (MVA) pathway, located in the cytosol, although with some emerging exceptions and crossovers. The MVA pathway is responsible for the production of the precursors of various terpenes and specifically the sesquiterpenes from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), while the MEP pathway provides these precursors for the formation of hemiterpenes, monoterpenes, diterpenes and isoprenes, which are precursors for the prenylation step towards a variety of phytoalexins, such as glyceollins (2 or C4 of the pterocarpan skeleton (glycinol). The contribution of the MVA pathway to glyceollin production occurs at the step catalyzed by isopentenyl diphosphate Δ-isomerase, which converts IPP into DMAPP, which can be transported from the cytoplasm to the chloroplast and elongate the pterocarpan skeleton formed from daidzein . Damage caused by rust in Brazil varies from 10% to 80%  is a disease caused by the obligate biotrophic fungus Rpp genes can trigger resistance responses related to some metabolic pathways, and compounds formed by these pathways are involved in defense mechanisms  leaves was dependent on the mixture of three compounds . This blend was able to promote development of the pathogen 24 hours after inoculation (hai). However, the terpenoid farnesyl acetate, when separately evaluated, negatively regulated haustorial development, by suppressing differentiation of the fungal cells, and reducing 98% of colony development. However, it is unknown whether this phenotype was caused by induction of soybean defense mechanisms by VOC or whether the compound itself has an antifungal effect , involving the precursor daidzein and dimethylallyl diphosphate (DMAPP), as well as the action of the G4DT enzyme. P. pachyrhizi interaction. Additionally, they observed that this phenomenon was mainly present in genotypes containing resistance genes.Studies in soybeans have demonstrated that induction of glyceollin synthesis is a defense response of different types of stress -α-farnesene were detected in the initial hours after P. pachyrhizi inoculation, either in compatible (genotype Embrapa 48) or incompatible (PI 230970) interactions of the soybean with rust. The levels of this VOC were inversely proportional to the plant symptoms; namely, the more evident the infection success was, the lower the VOC production, validating the expression profile observed in these soybean accessions and revealing it as a potential marker associated with of rust disease symptoms on soybean.In the present study, genes involved in the cytoplasmic pathway for IPP production and genes encoding different prenylases involved in the production of mono-, di-, and sesquiterpenes and glyceollin synthesis were evaluated regarding their response to different soybean pathogens by P. pachyrhizi studies were used as selection criteria when different gene models were available for same enzyme annotation  -α-farnesene synthase (GmTPS) (E.C. 4.2.3.46) and glyceollin synthase (E.C. 2.5.1.36) were also considered.Seven genes encoding to the enzymes of the cytoplasmic MVA pathway acetyl-CoA acetyltransferase (E.C. 2.3.1.9), hydroxymethylglutaryl-CoA synthase (E.C. 2.3.3.10), mevalonate kinase (E.C. 2.7.1.36), phosphomevalonate kinase (E.C. 2.7.4.2), diphosphomevalonate decarboxylase (E.C. 4.1.1.33), and isopentenyl-diphosphate Δ-isomerase (E.C. 5.3.3.2) as well as four genes corresponding to the prenylases geranyl diphosphate synthase (E.C. 2.5.1.1), -farnesyl-diphosphate synthetase (E.C. 2.5.1.10), and geranyl-geranyl diphosphate synthase (E.C. 2.5.1.29) were include in this study. Likewise, genes encoding the terpene synthase  gene . The plants were grown in a greenhouse under controlled conditions of temperature (22 ? 1 °C), humidity (> 60%) and a photoperiod (12 h). After reaching V2 stage . The second trifoliate from each plant was collected at 12, 24, 48, 72, 96 and 192 hours after inoculation (hai) and immediately transferred to liquid nitrogen and stored at −80 °C.Soybean seeds of the cv Williams 82 (susceptible – W82) and the plant introductions (PIs) containing different V2 stage , the soyP. pachyrhizi-infected or uninfected frozen leaves using an RNA extraction kit with TRIzol® reagent . Analysis to RNA quantifying and qualifying were performed with a Uniscience NanoDrop ND-1000 spectrophotometer , at a wavelength of 230 nm, and via agarose gel electrophoresis, respectively. The RNA samples were treated with deoxyribonuclease I . The SuperScriptTM III Kit (Invitrogen) was used to synthesize cDNA from treated RNA according to the manufacturer's instructions, and, after, samples were stored at −20 °C. Validation of cDNA quality was performed using PCR with primers designed to anneal to two different exons of the soybean β-actin gene , to avoid DNA contamination.Each sample (100 mg per replicate) was separately ground with a pestle, mortar and liquid nitrogen. After, the samples were distributed into 1.5 mL microtubes and stored at −80 °C. Total RNA (~1 g) was isolated from P. pachyrhizi infection were evaluated using RT-qPCR. The enzymes were selected using KEGG, and complete sequences of the gene models were obtained from Phytozome[2] and used to design primers. Specific primers to each of the 13 gene models were designed using the software Primer3Plus[3] and Vector NTI AdvanceTM (Invitrogen). The sequences of the primers are listed in Table S1. A schematic pathway composed of all the genes selected for the analysis is presented in The expression profiles of the gene models involved in the biosynthesis of IPP (MVA pathway), terpenes and glyceollins during (-1/slope) formula was employed to calculate the reaction efficiency and to adjust the final primer concentration and the calibration curve was established based on the Ct and the log of the cDNA dilutions. All amplification reactions were performed in triplicate using the 7900 Real Time System Thermocycler (Applied Biosystems), according to the manufacturer's instructions.The cDNA samples were amplified with specific primers to each gene model at a final concentration of 0.1–0.5 μM using 1X Kit Platinum® SYBR® Green qPCR SuperMix UDG (Invitrogen–Life Technologies), according to the manufacturer's instructions. The β-actin gene was used as an endogenous control. The E = 10http://jtreeview.sourceforge.net, respectively).After initial steps at 50 ºC for 2 min (UNG activity) and 95 ºC for 10 min (activation of the AmpliTaq Gold polymerase), a two-step program of 95 ºC for 15 s and 62 ºC for 1 min was run for 40 cycles. Dissociation curves were obtained to guarantee the absence of nonspecific amplification. The data were collected in the log phase, and the results were analyzed with the Sequence Detection program . The final relative quantification of each gene compared with the control was estimated considering the relative quantification (RQ) obtained in each biological replicate, as represented by each independent experiment, with three replicates each. Significant differences were defined based on estimates of the Relative Expression Software Tool (REST) version 2.0.7, with a significance level of 5%. Additionally, expression levels were converted to a log2-based colorimetric scale and represented as a heatmap using the software Cluster and Tree View  using an AP59 vortex mixer (Phoenix Luterco®) until completely homogenized. After sonication (20 min) (ultrasonic cleaner LS-Logen), the samples were centrifuged  and filtered through a Millipore filter (Ø = 0.22 μm).−1, and a mobile phase composed by MeOH and ultrapure H2O. The gradient flow was: 0-8 min [H2O/MeOH60:40], 8.5 min , 14.5 min , 15 min of [MeOH100%], 15.5-17 min, . An Acquity UPLC BEH C18 column  was used. Data were taken using Masslynx V4.1 software (Waters®).The ultrafiltered samples were analyzed by liquid chromatography with data photo diode array detector (UHPLC-PDA) and mass detection (Xevo Q-Tof) (both devices manufactured by Waters®) at the Chemical Ecology Laboratory of Embrapa Soybean, Londrina, PR. Chromatographic reversed-phase conditions were performed as follows: a sample injection volume of 10 μL, a run time of 17 min, a flow rate of 0.3 mL minAdditionally, using leaf samples of Embrapa 48 and accession PI230970 of three-week-old were analyzed for identification of the Volatile Organic Compounds (VOC's). These plants were maintained in glass container , opened at the bottom and with two collection openings, one lateral for air entry and one at the top (for outlet) to VOC acquisition according the standard procedure of The samples were analyzed by gas chromatography (GC) (Agilent 6890). Conditions were as follows: The oven temperature was maintained at 30 °C for 1 min, and set at 5 °C/min to 150 °C, and maintained for 0.1 min, and then 10 °C/min to 250 °C. Four microliters of each eluted sample were injected into the GC. Data were analyzed using HP ChemStation software. The quantification of VOCs was standard samples .in silico analysis of soybean public transcriptome data available for 13 model genes identified in the MVA pathway (Table S3), aiming to infer the involvement of genes related to terpenes production, derivated by the MVA pathway, in soybean defense mechanisms in response to different biotic stresses, in particular by P. pachyrhizi. The transcriptome analysis allowed the identification of nine studies, where at least one of 13 enzymes was differentially expressed, in at least one treatment in response to different pathogens, in at least three studies of soybean responses to infection with P. pachyrhizi (Table S3).We conducted an Based on transcriptome data, the main enzymes of the MVA pathway had the same expression profile for most interactions. For example, up-regulation of isopentenyl-diphosphate Δ-isomerase and geranyl diphosphate synthase and down-regulation of diphosphomevalonate decarboxylase (Glyma.10g426300) were observed.Rpp1, Rpp3 and Rpp4 genes, with only a subset of the enzymes -farnesyl-diphosphate synthetase and geranyl-geranyl diphosphate synthase) displaying down-regulation in the periods evaluated. From all significant expression changes, around 18% correspond to the P. pachyrhizi and soybean interaction, independent whether the interaction was compatible or incompatible . The levels of expression (fold-change) ranged from −95.14  to 34.66  -α-farnesene synthase). The differential expression of the genes encoding the enzymes mevalonate kinase, phosphomevalonate kinase,  farnesyl-diphosphate synthase and geranyl diphosphate synthase was detected in all three experiments involving soybean backgrounds containing the resistance genes. All significant expression changes are summarized in Table S3.It was possible to observe a differential expression of all 13 evaluated genes in at least one of the experiments involving the genotypes harboring H. glycines  -α-farnesene synthase (GmTPS) was not observed; instead, a strong downregulation of this gene was observed.According to the RT-qPCR analysis, in the susceptible genotype Williams 82 (W82), most of the genes that participate in IPP production were repressed or not differentially expressed . Only thRpp2 genotype, a slight up-regulation of the enzyme acetyl-CoA acetyltransferase was observed, while the other genes were repressed or not differentially expressed, with the exception of the later up-regulation of the enzyme isopentenyl-diphosphate Δ-isomerase. The prenylases enzymes geranyl diphosphate synthase and geranyl-geranyl diphosphate synthase were induced in 12 and 24 hai, respectively, even after significant down-regulation of genes encoding MVA pathway enzymes and, consequently, the IPP levels. There was considerable down-regulation of -farnesyl-diphosphate synthetase at all evaluated timepoints.In the Rpp4 genotype, slight up-regulation of hydroxymethylglutaryl-CoA synthase, mevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl-diphosphate Δ-isomerase was observed at specific timepoints. However, we observed up-regulation of prenylases involved in the formation of FPP precursors -farnesyl-diphosphate synthetase), followed by up-regulation of GmTPS  -α-farnesene synthase - E.C. 4.2.3.46). Significant down-regulation of geranyl-geranyl diphosphate synthase was observed. It was also observed up-regulation of glyceollin synthase involved in production of glyceollin at all timepoints studied.In the Rpp5 harboring genotype, differently from the other Rpp sources, the pathway was strongly induced, except for the enzyme hydroxymethylglutaryl-CoA synthase. The strongest up-regulation was observed in the enzymes phosphomevalonate kinase and diphosphomevalonate decarboxylase, which reached expression levels between 79.8 and 137 times more induced in inoculated samples compared to the false-inoculated samples at 48 hai. At the same timepoints, strong down-regulation of isopentenyl-diphosphate Δ-isomerase was observed, coinciding with the up-regulation of prenylases enzymes geranyl diphosphate synthase and -farnesyl-diphosphate synthetase. At 12 hai, the enzyme geranyl diphosphate synthase was upregulated in all genotypes, except in the Rpp5 genotype.In the E,E) α-farnesene (E.C. 4.2.3.46) was generally repressed after P. pachyrhizi infection, with the exception of the expression pattern observed in the Rpp4 background. All results can be found in Table S2.The enzyme glyceollin synthase was intensely induced at almost all timepoints and in all genotypes evaluated after inoculation with the fungus. However, GmTPS involved in the production of sesquiterpene  α-farnesene, that was later confirmed by a co-injection of the VOC solution and the external standard  α-farnesene. The peak obtained on the chromatogram was unique at the retention time corresponding to  α-farnesene.Comparing the spectra obtained with the spectral mass database, the reduced compound was identified (over 70% probability) as the metabolite  α-farnesene reduction among infected plants at the initial times of infection evaluated  α-farnesene in the infectious process. Leaves developed disease symptoms characterized as TAN lesions (Embrapa 48) and RB-type lesions (PI 230970), both in which solutions were added  α-farnesene as in used as control. However, further testing needs to be performed, especially regarding an efficient dilution of the compound (results not shown).Tests performed with detached soybean leaves inoculated with The MVA pathway consists of six enzymatic reactions initiated by the condensation of three acetyl-CoA units, forming 3-hydroxy-3-methylglutaryl-CoA, which is reduced to mevalonic acid, followed by two additional steps of phosphorylation and decarboxylation, culminating with the formation of IPP, the main precursor for the formation of terpenoids in the cytoplasm. Six genes encoding enzymes of the MVA pathway, culminate with the production of IPP precursor. The transcript levels of the enzyme isopentenyl diphosphate isomerase (IDI), which is related to the conversion of IPP to DMAPP, were also analyzed here, once DMAPP is an important precursor of glyceollin, by the action of the glyceollin synthase .E,6E)-farnesyl diphosphate synthase), responsible to convert IPP into GPP (geranyl diphosphate), FPP (farnesyl diphosphate) and GGPP (genranyl-geranyl diphosphate); and later by different terpene synthases, that use these precursors as substrates for monoterpenes (10 carbons), sesquiterpenes (15 carbons) and diterpenes (20 carbons) production. The enzyme GmTPS -α-farnesene synthase acts in the final step of  -α-farnesene (a sesquiterpene) production, converting FPP or GGPP into -α-farnesene  was observed in different soybean-pathogen interactions on the transcriptome studies, corroborating that these pathways are broadly activated by biotic stress in soybean, as part of the basal resistance response. Production of these compounds may not require specific recognition by the pathogen.P. pachyrhizi infection, but was not specific for this pathogen. The data also did not reveal a pattern of activation or down-regulation of a specific pathway in a specific pathosystem. We observed only five genes differentially expressed in response to a specific pathogen, including the enzymes acetyl-CoA acetyltransferase, diphosphomevalonate decarboxylase, geranyl diphosphate synthase, -farnesyl diphosphate synthase and geranyl-geranyl diphosphate synthase in response to aphids. Similarly, acetyl-CoA synthase was specifically upregulated in experiments where soybeans were infected with P. sojae  and in the Table S2).Most genes in the W82 genotype leading to the production of IPP were either repressed, and it was possible to observe a discrete up-regulation during haustorial development (48 hai) of three genes . At 192 hai, which corresponds to the uredia formation, four genes were induced . However, strong down-regulation was observed at 72 hai, where four genes  were significantly repressed, and the other genes were not differentially expressed, which is characteristic of a biphasic response, with two up-regulation peaks interspersed by a strong down-regulation of gene expression , returning to basal levels until 192 hai, when it was significantly induced again, coinciding with the penetration and sporulation stages of the P. pachyrhizi infection cycle, demonstrating that at least the formation of monoterpens precursors is common among the genotypes and its production might be associated with the initial and late steps of infection. The Rpp5 genotype was the only one where this biphasic pattern was not observed (Table S2).Interestingly, even though we could not observe a similar pattern in the MVA pathway among the different Table S2). It should be noted that, as in W82 genotype, the enzyme isopentenyl diphosphate isomerase, responsible for reversible reaction of IPP to DMAPP, was induced in Rpp4 genotype at the initial timepoints. This shows that even with slight production of IPP by the MVA pathway in these genotypes, the IPP may still be converted into DMAPP and, consequently, may be involved in other terpenoids production, such as mono- and diterpenes, as demonstrated by expression profile of different prenylases observed in this study in this genotype.The Rpp4 genotype likewise W82, displayed a slight up-regulation of four MVA pathway genes evaluated  at specific timepoints. A biphasic response was not observed  -α-farnesene synthase (GmTPS); instead, a strong down-regulation of this gene was observed (Table S2), suggesting that there is no  -α-farnesene production in compatible reactions, or in lower levels, corroborating with results of VOCs detection in susceptible genotype Embrapa 48 after rust infection -farnesyl diphosphate synthase) establishment was observed, followed by up-regulation of GmTPS  -α-farnesene synthase), indicating the possible production of , α-farnesene, at least during haustorial development and secretion of effector proteins (48 hai). Additionally, significant down-regulation of geranyl-geranyl diphosphate synthase was observed, indicating “capture” of the FPP precursor, favoring production of sesquiterpenes over diterpenes (Table S2). E,E) -α-farnesene synthase in a genotype containing the Rpp1 (PI200492) resistant-gene, after infection with two different isolates (HW94-1 and TW72-1) at the same timepoint of 48 hai (Table S2).In Rpp2 genotype displayed strong down-regulation of all MVA pathway genes evaluated in response to P. pachyrhizi. A slight up-regulation was only observed in first enzyme of the pathway (acetyl-CoA acetyltransferase) at 24 hai and at final timepoint evaluated (192 hai). Table S2). Repression of MVA pathway in Rpp2 genotype may compromise the production of IPP, the main precursor for terpenoid production in cytoplasm. This result was demonstrated by evaluation of the VOC sesquiterpene  α-farnesene production. This sesquiterpene was repressed after P. pachyrhizi infection in both, susceptible Embrapa 48 genotype and a resistant genotype containing Rpp2 gene -α-farnesene -farnesyl diphosphate synthase at all timepoints evaluated in this genotype suggests that mono- and diterpenes are being preferentially generated in relation to the development of . Interestingly, at 48 hai, a strong down-regulation of isopentenyl-diphosphate isomerase was also observed, suggesting a possible accumulation of IPP. At this time, the pathogen was able to establish plant tissue colonization with haustorium formation and, consequently, direct contact with effector secretion in plant cytoplasm. The strong up-regulation of MVA pathway suggested immediate detection of fungus by this genotype and the potential importance of terpenoid synthesis in resistance response mediated by Rpp5. The repression of the prenylase isopentenyl diphosphate isomerase at intermediate timepoints, coinciding with up-regulation of the enzymes geranyl diphosphate synthase and -farnesyl diphosphate synthase, indicated that mono- and diterpene synthesis is preferentially occurring in this genotype.Distinct from other genotypes evaluated in this study, the genotype containing the in silico and in vivo analyses, it is possible to conclude that there is a group of terpenes produced by plant depending on the type of biotic stress. In the case of soybean response to P. pachyrhizi, we observed a differential induction of genes involved in terpenes production dependent on Rpp genotype. However, we could not observe a significant difference between susceptible and resistant genotypes studied, indicating that -α-farnesene and other terpenes are involved in plants basal defenses, in general.Based on E,E)-α-farnesene production in plants is derived from FPP or GGPP precursors. The enzyme GmTPS -α-farnesene synthase acts in the final step of volatile α-farnesene, converting the precursors into -α-farnesene.The sesquiterpene -farnesyl diphosphate synthase was verified, followed by GmTPS down-regulation in Rpp2 genotype, confirming that in this genotype, the precursors generated were potentially used in diterpene synthesis, and subsequent  -α-farnesene reduction observed by VOC levels.A strong down-regulation of prenylase (2In the susceptible genotype (W82), although FPP had been synthesized, significant GmTPS down-regulation was observed, suggesting that in this genotype, FPP was used as a substrate for mono- and diterpene production.Rpp2 (PI230970) accession and in the susceptible genotype Embrapa 48, we could demonstrate the reduction of levels of the VOC  -α-farnesene across the infection cycle, corroborating with the expression profile of the genes in MVA pathway, the prenylases and GmTPS observed. Interestingly, the  -α-farnesene production was negatively associated with the P. pachyrhizi symptoms at least in these two genotypes. The reduction of the VOCs levels could be detected before the symptoms appearance.In the E,E) α-farnesene may be produced at least at specific time of 48 hai in this genotype. Additionally, the down-regulation of the enzyme geranyl-geranyl diphosphate synthase may be a strategy to save the prenyl precursor FPP, repressing diterpene production and contributing to sesquiterpene production.GmTPS up-regulation was only observed in Rpp4 background and was coordinated with prenylases activation involved in FPP formation, indicating that the VOC -farnesyl diphosphate synthase and no activation of GmTPS, indicated that mono- and diterpene synthesis might be preferentially occurring in this genotype.E,E) α-farnesene in these genotypes (Rpp4 and Rpp5) are necessary to confirm the expression profile results.Further studies to determine the levels of the VOC (Table S2), independent of which prenylases associated with different classes of terpenes were preferentially induced. Induction of glyceollin production has been reported in soybean in response to infection by P. pachyrhizi -glycinol 4-dimethylallyltransferase (G4DT), was dramatically induced after fungal inoculation at almost all time points and in all genotypes evaluated . The glyceollin level was detected from 24 hai, culminating with the peak area at 192 hai . These results confirm the importance the glyceollins in basal plant defense mechanisms . It is possible that the gene down-regulation observed in these genotypes is a plant mechanism to concentrate energy expenditure to ensure glyceollin production, an important phytoalexin in soybean with great influence in host defense reactions. Similar observations in this study, the in silico analysis found that the enzyme isopentenyl diphosphate isomerase was induced at all timepoints and stresses studied, indicating the importance of this enzyme in plant defense mechanisms and connecting IPP production between the cytoplasm and plastids, and providing the cytoplasm with alternative source of IPP.The down-regulation of some genes encoding MVA pathway enzymes observed in the W82, P. pachyrhizi infection in soybean. Even though, when compared to mRNA levels, protein elevation displays a slower response after detection.Based on the observed transcript expression profile and glyceollin levels peak, it is possible to suggest that this phytoalexin has an important role in basal resistance response to


Chenopodium album, Plantago lanceolata and Eucalyptus globulus were added to a confluent monolayer of Calu-3 cells grown in an air-liquid interface system. We identified serine proteases and metalloproteinases in all pollen diffusates investigated. Proteases found in these pollen diffusates were shown to compromise the integrity of the lung epithelial barrier by disrupting transmembrane adhesion proteins E-cadherin, claudin-1 and Occludin, as well as, the cytosolic complex zonula occludens-1 (ZO-1) resulting in a time-dependent increase in transepithelial permeability. Tight junction disruption and increased transepithelial permeability facilitates allergen exposure to epithelial sub-layers contributing to the sensitization to a wide range of allergens. These pollen extracts also induced an increase in the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) cytokines measured by flow cytometry possibly as a result of the activation of protease-activated receptors 2 (PAR-2).Allergic diseases are a major health concern worldwide. Pollens are important triggers for allergic rhinitis, conjunctivitis and asthma. Proteases released upon pollen grain hydration appear to play a major role in the typical immunological and inflammatory responses that occur in patients with allergic disorders. In this study, we aimed to identify specific proteolytic activity in a set of pollens with diverse allergenic potential. Diffusates from  Betula alba, Alnus incana, Corylus avellana, Platanus vulgaris, Cupressus sempervirens, Olea europaea, Artemisia vulgaris, Ambrosia artemisiifolia and Parietaria Judaica i in Calu-3 cells. Although both pollen diffusates induced an [Ca2+]i, increase, the response was different for each pollen diffusate. When exposed to C. album, the effect on [Ca2+]i was higher, and the cells were unable to return to basal conditions suggesting that C. album induced cell detachment even after exposure for short periods of time. In the present study, stimulation with 2+ mobilization and PAR activation, our results point toward a pathway in which serine and/or cysteine pollen proteases activate PAR-2 in the airways and induce IL-6 and IL-8 release. These results are consistent with those obtained with house dust mite and fungi extracts, which contain serine proteases and have been shown to directly induce airway inflammation characterized by IL-6 and IL-8 release in the airway epithelium i were evaluated by quantifying the ratio of the fluorescence emitted by Fura-2 at 510 nm after excitation at 340 and 380 nm. The acquired values were processed using the MetaFluor software . As PAR-2 is selectively activated by trypsin and trypsin-like enzymes, the effect of a trypsin solution (0.25%) on [Ca2+]i was used as a positive control.Calu-3 cells were grown on 12 mm glass coverslips and maintained in culture until confluency. Cells were loaded with 5 µM Fura-2/AM , 0.1% fatty acid-free BSA and 0.02% pluronic acid F-127 (Molecular Probes) in Krebs solution with calcium  according to the manufacturer’s instructions. The assay is based on a mixture of beads of different sizes that are coated with capture antibodies specific for each cytokine to be analyzed. A biotin-conjugated secondary antibody mixture was used to detect the cytokine of interest. After washing to remove unbound antibody, a streptavidin-phycoerythrin reagent was added. After removal of unbound material by washing, the bead suspension was analyzed using a FACSCalibur flow cytometer . Bead size and bead fluorescence identify the type of cytokine, and phycoerythrin fluorescence was used as a measure of cytokine concentration based on standards of known concentrations. The results were analyzed using the FlowCytomix Pro 2.4 Software (Bender MedSystem GMbH) and expressed in pg/ml for each cytokine. p-values were <0.05. The results are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-test (inhibition assays) or two-way ANOVA followed by Bonferroni´s post-test . Differences were considered significant when C. album, P. lanceolata and E. globulus release proteases with a significant in vitro effect on the paracellular permeability of epithelial cells and induced major degradation of cell junction proteins: E-cadherin, claudin-1, occludin and ZO-1. Moreover, our data suggests that the loss of effectiveness of the epithelium barrier may also occur via activation of PAR-2 followed by an immune response characterized by an increase in the release of IL-6 and IL-8.This work demonstrated for the first time that pollen diffusates from


Pulmonary embolism (PE) is a common and life-threatening medical condition with non-specific clinical presentation. Computed tomography pulmonary angiography (CT-PA) has been the diagnostic modality of choice, but its use is not without risks. Clinical decision rules have been established for the use of diagnostic modalities for patients with suspected PE. This study aims to assess the adherence of physicians to the diagnostic algorithms and rules.A retrospective observational study examining the utilization of CT-PA in the Emergency Department of King Fahd Hospital of Imam Abdulrahman Bin Faisal University for patients with suspected PE from May 2016 to December 2019. The electronic health records were used to collect the data, including background demographic data, clinical presentation, triage vital signs, D-dimer level (if ordered), risk factors for PE, and the CT-PA findings. The Wells score and pulmonary embolism rule-out (PERC) criteria were calculated retrospectively without knowledge of the results of D-dimer and the CT-PA.n = 114) or with normal D-dimer level (n = 5) despite being in the “PE unlikely” group. Only 49 patients had negative PERC criteria, of which three patients had PE.The study involved a total of 353 patients (125 men and 228 women) with a mean age of 46.7 ± 18.4 years. Overall, 200 patients (56.7%) were classified into the “PE unlikely” group and 153 patients (43.3%) in the “PE likely” group as per Wells criteria. Out of all the CT-PA, 119 CT-PA (33.7%) were requested without D-dimer assay (The study revealed that approximately one-third of all CT-PA requests were not adhering to the clinical decision rules with a significant underutilization of D-dimer assay in such patients. To reduce overutilization of imaging, planned interventions to promote the adherence to the current guidelines seem imperative. Pulmonary embolism (PE) is a common and potentially life-threatening medical condition. Every year, there are approximately 100,000 deaths from PE in the USA . Early dDiagnostic algorithms and clinical decision rules have been established to guide in the management of patients with suspected PE. Such criteria include the Wells criteria and the pulmonary embolism rule-out criteria (PERC) which were found to have high predictive accuracy , 10. In In Saudi Arabia, however, there has been no previous study investigating the adherence of physicians to the clinical decision rules for patients with suspected PE. Therefore, this study was designed to assess the adherence to these rules in the emergency department (ED).We conducted a retrospective study examining the adherence of physicians to the clinical decision rules for PE among patients with suspected PE in the ED.The study was conducted in King Fahd Hospital of the Imam Abdulrahman Bin Faisal University, which is a 440-bed capacity academic center and is located in the Eastern Province of Saudi Arabia. Approximately 250,000 patients visit the ED and 20,000 patients are admitted every year. In our institution, CT-PA can only be requested by board-certified consultant physicians.Eligible patients included all adults (≥ 18 years) with suspected PE who underwent CT-PA. The radiology information system was utilized to identify all CT-PA requests from the ED from May 2016 to December 2019.A web-based structured data collection form was used to collect the data from the electronic health record. The collected data included background demographic data, clinical presentation, D-dimer level (if ordered), risk factors for PE , and the CT-PA findings.The imaging results were collected from the electronic reports for assessing the presence of pulmonary artery filling defects. In case the patient had more than one CT-PA scan, only the most recent was included in our study. The Wells and PERC scores were calculated retrospectively by investigators without the knowledge of D-dimer and CT-PA results. The patients were categorized into the “PE likely” group if the Wells score was > 4 and the “PE unlikely” group if the Wells score was ≤ 4.The dual-source 128-slice scanner  with the administration of 25-ml non-ionic contrast material with timed intravenous pump infusion followed by a 25-ml saline flush, after the test bolus.The 64-slice scanner  with the administration of 60-ml non-ionic contrast material with timed intravenous pump infusion followed by a 40-ml saline flush, with bolus tracking.The scans were performed from the diaphragm to the lung apices, using one of the two multidetector scanners:A rapid quantitative immunoturbidimetric assay  was used for the D-dimer assay. The results were expressed as mg/ml and the normal range was defined as < 500 mg/ml. The result was usually reported within 1 h.α = 0.05.The obtained data were compiled using the QuestionPro platform and analyzed using IBM SPSS for Windows, version 25 . Continuous variables were presented as mean ± standard deviation. Descriptive statistics, such as percentages and frequency distribution of different characteristics, were used as appropriate. Categorical variables were compared using chi-square test. The statements of statistical significance were based on a significance level of The study involved a total of 353 patients, including 125 men and 228 women, representing 35.4% and 64.6% of the study population, respectively. The mean age of patients was 46.7 ± 18.4 years .Based on Wells criteria, 200 patients (56.7%) were classified into the “PE unlikely” group and 153 patients (43.3%) in the “PE likely” group  had PE including 15 men and 14 women. In 63 patients (41.2%), the CT-PA was performed following D-dimer assay. The D-dimer assay was elevated in all these patients, except for one patient who had normal level and subsequently had a normal CT-PA result.n = 114) or with normal D-dimer level (n = 5) despite being in the “PE unlikely” group.Among the “PE unlikely” group, the D-dimer was ordered in 86 patients (43%) only. Thus, there were 114 patients (57%) in the “PE unlikely” group who had CT-PA as the first investigation for PE. Of the 114 patients, 88 patients (77.2%) had a negative CT-PA. The CT-PA was performed despite a negative D-dimer for 5 patients from the “PE unlikely” group and none of them had a CT-PA positive for PE. Out of all the CT-PA, 119 CT-PA (33.7%) were requested without D-dimer assay (There were 49 patients (13.9%) having negative PERC criteria Table . Only thOur study revealed that one-third of all CT-PA performed in our institution were not in line with the clinical decision rules. This is because the CT-PA had been requested without having a positive D-dimer level despite the patient being in the “PE unlikely” group. Because the Wells score includes a subjective item which is “an alternative diagnosis is less likely than PE” that has a large contribution of three points to the score, it was initially proposed that the high proportion of unjustified CT-PA scans may be attributed to the retrospective nature of the study. However, we calculated the Wells score again with the assignment of these three points to all patients. Interestingly, the analysis showed that the D-dimer level was not ordered in 18.7% of patients despite being advised by the decision rules, thus, confirming our initial finding.The reason behind the underutilization of the D-dimer assay is unclear, but the increased patient load and time constraints in the ED might play a role. Furthermore, the use of CT-PA is often justified due to the finding of alternative diagnoses explaining the patient’s clinical presentation, though some evidence did not support this argument , 15. An Additionally, we had five cases (1.4%) in the “PE unlikely” group who underwent CT-PA despite a normal D-dimer level. Although this seems a very low rate, these were unjustified given the high sensitivity of D-dimer assay which reaches 99% . NotablyThe finding of inadequate adherence to clinical decision rules is not unique. Several previous studies from different institutions showed comparable results –19. For We identified that 6.1% of patients had PE despite having negative PERC criteria. The PERC criteria were designed to identify patients in whom the risk of unnecessary workup outweighs the risk of PE. A prospective study involving 8138 patients with negative PERC criteria showed that less than 1% had PE . Our finThe CT-PA remains the first-choice diagnostic modality for the diagnosis of PE because of its high accuracy, especially when incorporated into the clinical decision rules . HoweverDifferent strategies have been proposed to increase adherence to the clinical decision rules in the diagnosis of PE. These include the clinical decision support interventions which are based on a computerized physician order entry that prompts the physician to use the Wells criteria when ordering a CT-PA scan. Clinical performance and feedback reports to physicians about their practice and outcome are another intervention to improve the utilization of CT-PA. Formal educational interventions with training sessions have been advised. It has been suggested that the combination of these interventions may have a more significant impact .This study is the first to investigate the utilization of CT-PA in Saudi Arabia. However, the present study has some limitations. The retrospective observational nature of the study is an important limitation. The radiological images were not reviewed for the confirmation of the reported findings. Also, the study did not involve the patients with suspected PE who had not undergone CT-PA. Lastly, the complete reliance on the radiology report for the diagnosis of PE is another limitation given that some studies had shown some concern about the overdiagnosis of clinically insignificant PE .The study revealed that one-third of all CT-PA was not adhering to the clinical decision rules with significant underutilization of D-dimer assay when indicated. Further studies are needed to explore the barriers leading to inadequate adherence to these rules. To reduce overutilization of imaging, planned interventions to promote the adherence to the current guidelines seem imperative.


Acrylamide is a toxic chemical substance produced when starch-rich foods are fried at high temperatures. Asthma is a chronic and complicated respiratory disease, of which genetic and environmental factors are the main triggers. Orally-received components may have an effect on asthma pathophysiology. The aim of this study was to investigate the role of AA as a stimulus in asthma. BALB/c mice were allocated into four groups as follows: two OVA-sensitized asthmatic groups, including one treated with AA by gavage feeding and one non-treated (asthma group), and two healthy (non-asthmatic) groups, one treated with AA by gavage feeding and one non-treated (negative control group). Airway hyperresponsiveness, cell count, cytokine levels in BAL fluid, lung histopathology, IgE levels, and oxidative stress indices including plasma level of MDA, pulmonary antioxidant enzymes (SOD and CAT) levels, HP content, and collagen fiber accumulation in lung tissue were measured. We found that the group of mice treated with both OVA and AA (asthmatic and AA-treated mice) experienced higher levels of asthma-associated biomarkers, including higher enhanced pause , eosinophilic inflammation, mucus hyper secretion, goblet cell hyperplasia, total and OVA-specific IgE levels, IL-4, IL-5, and IL-13 levels than the group sensitized only with OVA (asthmatic mice). The OVA-AA-treated mice also experienced worsened levels of oxidative stress indicators. Healthy (non-asthmatic) mice that only received AA were in similar conditions to healthy untreated mice (negative control group). The OVA-AA-treated group showed more severe allergic asthma symptoms in comparison to the group only sensitized with OVA. Therefore, food/water contaminated with AA can act as a stimulant of allergic asthma and exacerbate the bronchial inflammatory responses. Acrylamide is a toxic chemical substance found in baked and fried foods such as potato chips, cookies, and biscuits. When starch-rich foods are fried at high temperatures, AA is formed in the food. Asparagine is one of the main precursors of acrylamide formation . AA is fAA has toxicity effects on tissues and causes irritation of the skin, eyes, and airways. AA toxicity results from dermal absorption rather than from inhalation routes –6. PerviAsthma is one of the main complex syndromes of the respiratory system and is characterized by recurrent episodes of coughing, dyspnea, chronic airway narrowing, wheezing, increased bronchial responsiveness to inhaled stimuli, airway eosinophilic inflammation, mucus hyper secretion, and recurrent smooth muscle spasm. Asthma is a multifactorial disease controlled by genetic and environmental factors , 14. AllSix-week-female BALB/c mice (with a mean body weight of 19 g) were purchased from the Pasteur Institute  and acclimatized to the standard conditions  by being maintained in the animal room for 1 week before starting the experiments.n = 24). In two groups, airway inflammation was induced by ovalbumin  (OVA-sensitized) according to a previously described protocol  was measured by methacholine challenge a day before each instance of sacrificing the mice  in the manner described earlier –22. AHR The number of two WBCs  in the BAL fluid were calculated as the percentage's values. In the other types of white blood cells, the neutrophil is important to determine the presence of infection in studied animals. The increased percentage of neutrophils show that there is infection and bronchial inflammation was initiated by infection, not by allergic reactions, thereby making the produced models worthless. The levels of IL-4, IL-5, IL-13, and INF-γ in BAL fluid were measured using Multi (bio)-Plex Pro™ Mouse Cytokine, Chemokine, and Growth Factor Assays  as described before .Before sacrificing the mice, blood samples were collected from the tail vein and the percentage of Eos was determined. Blood serum was separated and then total and OVA-specific IgE levels in serum were measured by ELISA, anti-Mouse IgE ELISA kit .2O2 and then stopping the reaction by adding the AM. AM is a colorimetric indicator of the remaining H2O2 (it colors it to yellow) and the amount of H2O2 itself is directly associated with CAT degrading activity; the enzyme converts H2O2 to H2O and O2. The absorption of CAT experiments was read at 410 nm and the results were expressed as μmol H2O2/min/mg protein.The oxidative stress indicators, including plasma level of MDA, pulmonary levels of superoxide dismutase (SOD), and catalase (CAT), were measured using the lung tissues. SOD level was calculated from the absorption reads at 412 nm for samples from the lung tissue (Thermofisher. USA), and the results were expressed as IU/mg protein. CAT content was evaluated by a colorimetric method  which, as a brief explanation, was performed by incubating the lung tissue with HThe hydroxyproline (HP) content in the lung tissue, as an important index of the deposition of collagen fibers, was measured by a colorimetric modified method introduced by Woessner . Lung tiAt the end of each 2 weeks of AA-treatment period, the lung tissues of mice were isolated and maintained in formalin solution until fixation, then trimmed and paraffin-embedded. The sections were stained with Hematoxylin and Eosin (H&E), Alcian Blue, Alcian Blue-Periodic acid Schiff (PAS)-H&E, and Trichrome Masson stain. Afterwards, the perivascular and peribronchiolar inflammation were evaluated by using a routine scoring system . Goblet t-test was used to analyze AHR methacholine changes from the baseline. The differences between asthmatic groups (AA-treated and non-treated) and control groups and the differences between AA–treated groups (asthmatic and non-asthmatic) and non-treated groups (not OVA- or AA-treated) were analyzed using unpaired Student's t-tests. Multiple comparisons were done using one-way ANOVA followed by Tukey's HSD tests. The p value at the level of <0.05 was supposed to be significant. SPSS (ver. 18) has been used for analysis and analyses were performed using GraphPad Prism (version 5.0).All experiments were repeated three times and results have been reported as a mean of three times. Data were expressed as means ± standard deviations (SD). Correlation analysis was carried out using Pearson's method. The paired This study was approved by the institutional review board (code number: 425/641).p < 0.05) (p = 0.040). OVA-AA-treated group were significant different to OVA-sensitized and Penh values in the OVA-AA-treated group were higher than the asthmatic group in all concentrations. Penh value in healthy (non-asthmatic) AA-treated mice (at 4 mg/ml = 2.6 ± 0.08) were close to the levels in healthy untreated mice (at 4 mg/ml = 2.1 ± 0.10) indicating no significant change (p = 0.230). Other doses of MCh had similar patterns of 4 mg/ml, and all other paired groups had no significant differences between each other.The Penh values were significantly increased at all concentrations of MCh in the asthmatic mice compared to the negative control mice ( < 0.05) . Penh vap = 0.880). However, the number of Eos in BAL samples from OVA-AA-treated groups were significantly different compared to the OVA-only treated (asthmatic) group (p = 0.019) (p = 0.710). In all groups, the percentage of neutrophils in the BAL fluid were <4%, which shows that there was no infection in the studied animals and bronchial inflammation was produced by allergic reactions.Eosinophils in BAL and blood slides were counted. The number of Eos in blood samples from OVA-AA-treated groups were not significantly different compared to the OVA-only treated (asthmatic) group (= 0.019) , 4. In tp = 0.047, p = 0.049, respectively), at day 86. IgE levels in healthy (non-asthmatic) but not AA-treated group were close to the levels in the healthy untreated group and there was no significant difference between healthy and healthy AA-treated groups at day 86  compared to OVA-sensitized but not AA-treated group (asthmatic group)  (t day 86 .p < 0.05) . The levels of IL-4, IL-5, IL-13, and IFN-γ had no significant difference between the healthy AA-received group and the healthy group , whereas a reverse trend was observed for IFN-γ levels ( < 0.05) . The levhy group .p < 0.05) (23.00 ± 2.45 IU/mg protein), and it was decreased in the asthma AA-received group. Similarly, the CAT after 2 weeks had no significant difference between the asthma group (78.0 ± 5.35 μmol H2O2/min/mg protein) and asthma AA-received group (77.7 ± 4.11 μmol H2O2/min/mg protein). At week 8, CAT showed a significant difference between the asthma group and asthma AA-received group and it was decreased in the asthma AA-received group  (p < 0.05). In the 2nd week, MDA in the asthma AA-received group (2.2 ± 0.36) was increased significantly when compared with the asthma group (2 ± 0.24) and In the 8th week (p < 0.05), MDA was increased significantly in the asthma AA-received group compare with the asthma group (p < 0.05)  . But at 8 weeks, SOD levels had a significant difference between the asthma group (44.33 ± 2.87 IU/mg protein) and asthma AA-received group (ctively) .p < 0.05)   .p < 0.05). Mucus hyper secretion and goblet cell hyperplasia were significantly induced in the airways of OVA-challenged mice that also received AA (asthma AA-received group) compared to the group only treated by OVA (asthmatic group) (p < 0.048) (p < 0.018). Inflammation in the PBS-challenged AA-treated group was at a similar level to that in the PBS-challenged but untreated group  compared to PBS-challenged mice  (< 0.048) , 10. Hised group , 10.50 of AA was reported at 107 mg/kg body weight (b.w.) for mice (10) was reported at 0.43 mg/kg b.w. per day for peripheral neuropathy in rats and at 0.17 mg/kg b.w. per day for neoplastic effects in mice . The symptoms seen in the non-asthmatic but AA-treated group was at a similar level to that seen in the healthy untreated group.Acrylamide is toxic for cells and has a poisoning effect on tissues. It is used industrially as a monomer precursor for the production of dyes and polymers such as sodium dodecyl sulfate polyacrylamide gel , 28. AA AA is found mainly in vegetarian foods and potato chips, which contain the highest levels of AA. Bread also contains high levels of AA . In thisIn asthmatic mice, AHR  was increased compared to in negative control mice. The level of AHR was higher in the OVA-AA-treated group in comparison to the OVA-only treated group (asthma group). The non-asthmatic AA-treated group showed no change in AHR level compared to the healthy untreated group. These results showed that AA worsens the inflammatory responses in asthmatic mice, but has little or no effect in healthy mice. Previous studies showed that AA can act as an allergic agent and cause a cutaneous anaphylaxis reaction, atopic dermatitis, eczema, and allergic reactions –31. In aDeGrandchamp et al. , have foEos is the predominant cell in BAL fluid and blood of asthmatic patients. In our study, the Eos number in BAL and the blood of OVA- AA-treated groups were higher than that in OVA-sensitized groups. Moreover, we found that AA can induce severe eosinophilic inflammation in asthmatic airways. Zaidi et al. in 1994, showed that AA causes immunotoxicity and reduction of circulating blood lymphocytes . In addiAspergillus ochraceus that prevents AA toxicity  from their dietary regimen.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The studies involving animals were reviewed and approved by ethics committee of the Shahid Sadoughi University of Medical Sciences.BH participated in the design of the study. SMA assisted in the establishment of airway hyperresponsiveness test and statistical analysis. MA supervised the study. GV assisted in result analysis and drafting the manuscript. SSA participated in the design of the study, animal works, lab analysis, and drafting the manuscript. This study is the outcome of the postdoc program of the SSA, who is one of the authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obesity has previously been linked to inflammation. Here we investigated how plasma levels of six interleukins were related to body fat distribution.2), six interleukins were measured together with a DXA scan for determination of fat and lean mass. Also a whole-body magnetic resonance imaging (MRI) scan, in which fat content measurements were acquired in > 1 million voxels was performed. Interleukin levels were related to each of these voxels by the voxel-based technique “Imiomics” to create a 3D-view of how these measurements were related to size of each part of the body.In 321 subjects, all aged 50 years, in the population-based POEM study , as well to ectopic fat distribution. In women, IL-16 levels were weakly related to expansion of SAT in the upper part of the body, while on the contrary, IL-8 levels were related to a reduction of SAT volume.Of the six evaluated interleukins, plasma IL-1RA and IL-6 levels were related to the amount of adipose tissue in all parts of the body, while a diverse picture was seen for other interleukins, suggesting that different interleukins are related to fat distribution in different ways. This was mainly based on the observations that C-reactive protein (CPR) and interleukin-6 (IL-6) levels were increased in obesity https://gtexportal.org/home/), all of the proinflammatory interleukins evaluated in the present study were expressed in human adipose tissue, with generally higher expression in VAT compared to SAT.Adipose tissue macrophages (ATM) are a pivotal part of both SAT and VAT, and are key to the association between interleukins and body composition. As recently reviewed by Li et al. It was early suggested that abdominal obesity was the main driver of the relationship between obesity and inflammation Most studies investigating the role of inflammation in obesity have used CRP and/or IL-6. However, many other interleukins exist with important effect in the immune system, and circulating levels of also other interleukins, such as IL-8, IL-16, IL-18 or IL-1/IL-1RA have been associated with obesity Another facet of ectopic fat accumulation than VAT is increased liver fat. Although inflammation is a suggested feature of non-alcoholic fatty liver disease (NAFLD), data on circulating levels of interleukins in NAFLD are rather scarce, but increased levels of IL-6 and IL-8 (mean BMI 26–27 kg/m2). The hypothesis evaluated was that the six interleukins would be related to fat distribution in different ways.The aim of the present study is to perform a detailed investigation of the relationships between 6 different interleukins and fat distribution using measurements performed by both a dual-energy X-ray absorptiometry (DXA) scan and magnetic resonance imaging (MRI). In addition, we applied a new voxel-based 3D-technique, “Imiomics”, by which interleukin levels were related to each of the > 1 million voxels included in a whole-body MRI scan 22.1In the Prospective study on Obesity, Energy, and Metabolism (POEM), 50-year-old men and women were recruited from the general population in the municipality of Uppsala, Sweden. Public population registers were used and invitation was done in a random manner by mail The participants were examined after fasting overnight. The blood samples were drawn between 8 and 10 am. After centrifugation, plasma was frozen in −80C until protein analyses.Blood pressure was measured in the supine position after 30 min of rest by a mercury sphygmomanometer. Fasting blood glucose and lipids were measured by conventional techniques at the hospital clinical chemistry laboratory.The participants were asked about their exercise habits, which were graded into four groups; sedentary, only light exercise only (no sweat), exercise at least 0.5 h 1–2 times a week and exercise > 2 times a week.The circumferences of the waist and hip were measured at the umbilical and trochanter levels, respectively. Using these measurements, waist to hip ratio (WHR) was calculated. Fat and lean mass measurements were established using DXA. Whole-body (head to toe) MRI as well as dedicated imaging of pancreas and liver were performed on subjects who volunteered for this part of the study. The MRI scan was acquired on a separate day, not more than one month from the main study visit. The study in this paper includes a subset of the POEM cohort consisting of the 326 subjects for which the MRI registration was successful None of the subjects reported any cardiovascular disease. The prevalence of diabetes was low in the sample .2.2Using the proximity extension assay (PEA) technique, 92 proteins were measured by the OLINK CVD-1 chip . In the present study, we evaluated the 6 proinflammatory interleukins with a call-rate > 75%. Details on measurements, quality control etc have been given previously 2.3A Dual-energy X-ray absorptiometry scanner  was used to measure regional  and total body fat and lean mass. One experienced nurse performed all scans to minimize potential operator bias. The scans were all performed in the same room. Triple measurements in 15 subjects with repositioning were used to calculate the precision error of the DXA measurements. This approach for computing the precision error follows the recommendations by the International Society for Clinical Densitometry. The precision error was 1.5% and 1.0% for total fat and lean mass, respectively. Automatic edge detection was consistently employed in the analysis. In addition, all scans were carefully checked for errors. Manual correction was performed when necessary.2.4A 1.5 T clinical MR system  was used to acquire whole-body MRI images of the subjects in supine position. The acquisition was carried out with a continuously moving bed setup using the integrated body coil. A dedicated scan of the liver and the pancreas was also undertaken to enable detailed analysis of liver and pancreas fat content. For additional information about the imaging protocol, see The liver and pancreas fat quantification was performed by manual delineation of the volume of interest. The software ImageJ (version 1.45 s) was used for the delineation. As much as possible of the volume of interest was demarked by a trained operator. Tissue borders were avoided to limit partial volume effects. As measurements of tissue fat content, the median fat contents were usedQuantification of the VAT and SAT depots were performed by deforming manually defined depots to all other subjects. The deformation was done by utilizing the same image-registration method that was used for the Imiomics analysis, as described below. This process was done separately for male and female. Further processing of the deformed regions (thresholding operations) was done to remove voxels with fat content < 50%.2.5The Imiomics technique builds on image registration. In image registration, deformation field is computed and applied to deform a target image to match a (fixed) reference image. The deformation field defines a point-to-point correspondence between the reference and target images. These correspondences are used in Imiomics analyses to deform whole-body MRI images to a single reference whole-body MRI volume. Each point in all volume images in the cohort has a corresponding point in the reference coordinate system, i.e., the reference whole-body MRI volume. The voxel-wise statistical analysis procedure It is crucial that the image registration method is reliable in Imiomics analyses, and a tissue-specific handling of bone, lean tissue and adipose tissue is employed. The degree of deformation elasticity tends to differ across these different tissue types, when aligning two images. The tissue specific processing is implemented in the image registration process by performing deformations of the different tissues sequentially and applying tissue-specific registration parameters. In the Imiomics image registration method, the following three steps are carried out: 1) Piece-wise affine articulated registration of bone sections; 2) Water image registration with constraints on bone; 3) Fat images registration with constraints on bone and water. The image registration method has previously been presented and evaluated 2.6Initially, a correlation matrix based on Pearsońs correlation coefficient was created for the six interleukins.Thereafter, relationships between traditional measurements of anthropology and fat distribution and the six interleukins was evaluated by linear regression analysis. The relationships are presented as regression coefficients and were stratified by sex. Some variables  were ln-transformed to achieve a normal distribution. In order to improve the comparison between measurements of anthropology and fat distribution, these were transformed to a standard deviation-scale. The analyses were performed unadjusted, since all subjects were aged 50 years.STATA 16 was employed for the computations .For the Imiomics analyses, r-value maps were generated for all aprameters by using Spearman non-parametric correlation. MATLAB  was used for these calculations. The colormap jet was used to visualize the r-values. The colormap jet represents strong positive correlations (near one) as dark red and strong negative correlations (near minus one) as dark blue.The Imiomics results were analyzed visually. Only associations corresponding to P-values far below 0.05 visible in many image elements within a certain anatomical structure were reported. This procedure was used to minimize the number of false positives from the large number of tests performed.33.1As could be seen in No major differences in the relationships between the measured plasma interleukins were seen between men and women see .Table 1T3.2As could be seen in Neither IL-6R, nor IL-8 or IL-18, showed any consistent significant relationships vs the traditional measurements of body composition, while IL-16 showed relationships being of borderline significance  for most of the adipose tissue measurements in the sex-stratified analysis, except for liver fat. These relationships all showed p < 0.05 when adjusting for sex (instead of stratifying on sex).When deleting the 7 diabetic subjects and adjusting for exercise habits, essentially the same results as reported above were seen.3.3IL-1RAIn both men and women, IL-1RA levels were closely related to expansion of both SAT and VAT, as well as to expansion of liver size and the blood volume in the heart see . Of notiIL-6In both men and women, IL-6 levels were related to the size and lipid content of different adipose tissue compartments in a similar fashion as IL-1RA. The relationships were slightly less pronounced for IL6 when compared to IL-1RA.IL-6RAIn sharp contrast to the findings for IL-6, IL-6RA levels were mainly related to an expansion of SAT in the gluteus area, especially in the dorsal parts, in women. This association was not as strong as for IL-6. In males, not significant relationship between an expansion of SAT and IL6RA levels was seen. The lipid content was not significantly related to IL-6RA.IL-8In women, IL-8 levels were related to a reduction of SAT in the upper part of the body, as well as a reduction in skeletal muscle volume in the leg. In males, this was less evident. Also a reduction in lipid content in proportion to IL-8 levels was seen in women, but less so in men.IL-16In women, IL-16 levels were weakly related to expansion of SAT in the upper part of the body. In males, this was mainly seen at the umbilical level. The lipid content was not significantly related to IL-16.IL-18In women, IL-18 levels were mainly related to a reduction of the lung volume in the lower parts of the lungs. In males, this was less evident. The lipid content was not significantly related to IL-18.4The present study showed that plasma levels of IL-1RA and IL-6 were related to SAT in both the upper and lower part of the body, as well as to indices of ectopic fat distribution, such as VAT, liver and pancreatic fat. Neither IL-6R, nor IL-8 or IL-18, showed any consistent significant relationships vs the traditional measurements of body composition, while IL-16 showed relationships being of borderline significance. In women, IL-16 levels were weakly related to expansion of SAT in the upper part of the body, while on the contrary, IL-8 levels were related to a reduction of SAT volume. Thus, different interleukins were related to fat distribution in different ways.All of the four evaluated interleukins are considered to be proinflammatory, although their actions differ , in which dedicated brain MRI scans were performed and brain volume was evaluated by a dedicated software (p = 0.89 vs IL-18 and p = 0.56 vs IL1RA) The major strength of the present study is that we measured fat distribution by both traditional measurements, DXA and MRI, but also by the novel voxel-based Imiomics method, which gives a detailed 3D view of relationships between interleukin levels and body composition.In the present study, six interleukins (or their receptor antagonists) were chosen from the prespecified CVD-1 chip manufactured by OLINK. If would have been desirable to evaluate many more interleukins, like IL-1 beta, IL-33, IL-15, IL-17 or IL-10, but these other interleukins were unfortunately not included amongst the available proteins.A limitation is that our sample consists of Swedish individuals all aged 50, so the results have to be confirmed in other samples with other geographical, age and ethnical characteristics.In conclusion, of six evaluated interleukins, plasma IL-1RA and IL-6 levels were related to adipose tissue in all parts of the body, while a diverse picture was seen for other interleukins, suggesting that different interleukins are related to fat distribution in different ways.Robin Strand: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Joel Kullberg: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Håkan Ahlström: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing - original draft, Writing - review & editing. Lars Lind: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.Joel Kullberg and Håkan Ahlström are cofounders, co-owners of and part time employees of Antaros Medical AB, BioVenture Hub, Mölndal, Sweden. A patent application, P1318PC00, by Robin Strand, Joel Kullberg and Håkan Ahlström describing the image registration method used in this manuscript is currently under review. Antaros Medical is currently holding the rights to the patent application.


For mass production of liposomes, we designed a plastic micro-channel device on the basis of 5 μm of micro-nozzle array forming T-junction with 100 μm depth of micro-channel. A micro-channel unit for synthesizing liposomes consisted of two micro-nozzle arrays for mixing two solutions as well as delivery and recovery channels for supplying solutions and collecting liposome suspension. The number of micro-nozzles was approximately 2400 for a micro-channel unit, and seven units were applied independently on a micro-channel plate. The plastic micro-channel plate was injection-molded for mass production using a micro-channel stamper previously fabricated by UV lithography and nickel electroforming process. A plastic cover plate with seven pairs of inlet and outlet ports was machined by mechanical milling and drilling and was assembled with a micro-channel plate using a holder to form a liposome synthesizing device. Flow and mixing of solutions in the micro-channels were tested using colored water to check the micro-fluidic characteristics of the device. Finally, a L-α-phosphatidylcholine (SOY PC) liposome was synthesized using EtOH solution of SOY PC (95%) and saline (0.85% NaOH solution) to find that the liposomes were around 230 and 260 nm in diameter, depending on the flow rate of the lipid solution. Liposome is a spherical lipid bilayer  that canTo synthesize liposome, several bulk synthesis methods are used, such as ultrasonic emulsification , extrusiIn this study, a micro-channel device was designed on the basis of T-junction type of micro-channel array for scalable production of liposome. The micro-channel plate was injection-molded using thermoplastic for mass production, mechanical strength, and dimensional stability. The injection-molded micro-channel plate was sealed through mechanical assembly using a plastic cover plate with inlet and outlet ports. Using the micro-channel device, we synthesized L-α-phosphatidylcholine (SOY PC) liposomes; afterwards, their size and distribution were measured.The micro-channel device for synthesizing liposome consists of a micro-channel plate and a cover plate with inlet and outlet ports . A microAs shown in First, two parts of the liposome-synthesizing device, a micro-channel plate, and a cover plate, as shown in For injection molding of micro-channel plate, we fabricated a durable nickel stamper to be 0.5 mm in thickness and to have negative structure for micro-channel based on UV lithography process on silicon wafer and nickel electroforming process. The overall fabrication process for nickel stamper is shown in As shown in 2 was applied first for 1 s and then 800 kgf/cm2 for 2.5 s.After mounting the nickel stamper block on the wall of the mold cavity, as shown in As shown in For sealing micro-channels and making the micro-channel device work, we stacked the micro-channel plate and cover plate and inserted them between two halves of an aluminum holder assembled by bolting to press the plates tightly . As seenFor testing flow and mixing of fluids through micro-nozzle and channels, we used two types of water, one yellow and the other blue, for visualization of flow and mixing. The yellow water was supplied to the delivery channel through the inlet port for lipid solution   and saline (0.85% NaOH solution) were used to synthesize liposome using micro-channel device. A total of 0.775 g of granular SOY PC was mixed with 10 mL of EtOH for 3 h using a magnetic stirrer to produce 10 mM of SOY PC EtOH solution. This SOY PC solution was supplied to the micro-channel device through inlet port 1 at two different flow rates, 3 mL/h and 4.5 mL/h. Saline, 0.85% of NaOH solution, was supplied through inlet port 2 at a 30 mL/h flow rate . LiposomThe liposome suspension was heated up to 80 °C for 1 h so that it could be volatilized after being collected through the outlet port from recovery channel where the two solutions were mixed. The boiled liposome suspension was cooled down to room temperature and stored in a chamber at 5 °C for 20 h. NANOPHOX , a photon cross-correlation spectroscopy (PCCS), was used to analyze the size of the liposome in the suspension.The results for the measured size of the SOY PC liposomes synthesized by micro-channel device are shown in From the viewpoint of throughput, 30 mL/h of flow rate in this work is much higher than a flow rate less than 1 mL/h for typical single T-junction micro-channel . This inA high-throughput plastic micro-channel device for liposome synthesis was designed and fabricated by injection molding or mechanical machining depending on part and assembling the parts. The micro-channel plate, a core part of the liposome synthesizing device, was injection-molded with a cycle time of less than 30 s, which is fast enough for mass production. The micro-channel device showed that fluids flowed and mixed well through micro-nozzles and micro-channels. Using the micro-channel device, we synthesized SOY PC liposome by mixing EtOH solution of SOY PC and saline (0.85% NaOH solution). The size of the liposome varied depending on the flow rate conditions of solutions. In the case of 30 mL/h of flow rate for saline, the liposome diameter was 230 nm or 260 nm for 3.0 mL/h or 4.5 mL/h SOY PC solution, respectively. The size of each liposome sample had a standard deviation of less than 20 nm, which indicates good uniformity in size.Overall, this study implies that the throughput for synthesizing liposome can be enhanced linearly by using multiple micro-channel units. Additional extensive studies are required to figure out the precise correlation between size of the liposome and flow rate.


First, liposomes containing dexamethasone (Dex-L) are prepared and designed to entry into the macrophages in the early hours, forming the macrophages loaded Dex-L delivery system (Dex-L-MV). Chemokine and cytokine factors such as IL-6, IL-10, Arg-1 are measured to show the effect of Dex-L to the various subtypes of macrophages. Next, we mimic the inflammatory and anti-inflammatory microenvironment by co-culture of polarized/inactive macrophage and fibroblast cells to show the acute inflammation response of Dex-L-MV. Further, we confirm the targeted delivery of Dex-L-MV into the inflammatory sites in vivo, and surprisingly found that injected macrophage containing Dex can reduce the level of macrophage infiltration and expression of the markers of collagen deposition during the fibrotic stage, while causing little systematic toxicity. These data demonstrated the suitability and immune regulation effect of Dex-L-MV for the anti-pulmonary process. It is envisaged that these findings are a step forward toward endogenous immune targeting systems as a tool for clinical drug delivery.Idiopathic pulmonary fibrosis (IPF) is serious chronic lung disease with limited therapeutic approaches. Inflammation and immune disorders are considered as the main factors in the initiation and development of pulmonary fibrosis. Inspired by the key roles of macrophages during the processes of inflammation and immune disorders, here, we report a new method for direct drug delivery into the  Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia of unknown etiology. There is currently poor prognosis with increased morbidity and prevalence . CurrentThe strategy exploiting immune systems for drug delivery is an area of great interest, as immune cells such as macrophages, neutrophils, and dendritic cells could sensitively respond to the complex inflammatory microenvironment at acute injury sites . These wDepending on the local microenvironment on the fibrotic stage, macrophages can be replaced with classical activation (M1) and alternative activation (M2) phenotypes . In geneExploiting macrophage for drug delivery causing anti-inflammatory response has previously been demonstrated in tissues other than the lung. Zheng et al. used tungsten oxide (WO) and indocyanine green (ICG) to construct nanoparticles. After macrophages swallowed the nanoparticles, the cells were injected through the tail vein to make the cells reach the tumor site and then given near-infrared irradiation which kill the engulfed tumor cells by photothermal effect . Zhang eIt is worth noting that there are no reports of using macrophages as a delivery system for the treatment of pulmonary fibrosis. Moreover, no studies have reported the effect of drug-containing nanocarriers on the phenotypic changes of macrophages during the treatment of diseases. Here, we report a macrophage-mediated drug delivery system, which achieve efficient and accurate pulmonary drug delivery, with improved bioavailability of anti-pulmonary fibrosis drug dexamethasone (Dex) into the lung. Results showed that Dex decorated with liposomes (Dex-L) increased the loading efficiency of Dex into macrophages, similarly it had a bidirectional regulation effects to the phenotype when responding with the polarized macrophages which exacerbate the lung damage during the process of fibrosis. It was proved that Dex-L could keep the phenotype of the carrier itself in a balanced and non-pathogenic state, which also provided an explanation for the mechanism of Dex against pulmonary fibrosis. The therapeutic effects were further evaluated in a co-culture cell model of macrophages and fibroblasts and a mouse model of bleomycin-induced pulmonary fibrosis. We found that macrophages as a delivery system could effectively deliver Dex to the lungs with little systematic toxicity and exhibited excellent ability to inhibit pulmonary fibrosis. It is expected that this approach may provide an improved platform for targeted delivery of anti-fibrotic drugs while minimizing the problems of side effects.1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoyl phosphoethanolamine and (DOPE), cholesterol (CHO) were purchased from Avanti Lipid . All antibodies, including Alpha smooth muscle actin (α-SMA) mouse monoclonal, rat anti-F4/80 antibody, Rabbit anti-CD206 antibody, Alexa Fluor 488-conjugated goat anti-rabbit antibody, and Alexa Fluor 647-conjugated goat anti-rat antibody, were purchased from Abcam . BLM-A5 hydrochloride was purchased from Dalian Meilun Biotech . Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich . IL-4 was purchased from PeproTech . TGF-β1 was purchased from R&D system. The ELISA test kit for IL-6, TGF-β1, IL-10, IL-1β, and Arg-1 were purchased from Zhuo Cai Biological Co., Ltd. . Nitric oxide synthase test kit was purchased from Biyuntian Co., Ltd. . Masson's trichrome assay kit were purchased from Solarbio . Sirius red reagent were purchased from Yuanye Bio-Technology . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Solarbio . Dulbecco's Modified Eagle's Medium (DMEM) and other cell culture supplies were obtained from Gibco . Hoechst was provided by Invitrogen Co. (USA). Four percentage formalin was purchased from Weiao Biological .Dissolved DOPC , cholesterol and DOPE  in a molar ratio of 2:1:1 in a mixed solvent of chloroform-methanol  and dexamethasone dissolved in methanol with molar ratios of 1:1 in a 20 mL glass to form a film under nitrogen, respectively. Then the PBS  was added to form a lipid suspension and ultrasonicated in a water bath at temperature 55°C for 30–40 min.The diameter distribution and zeta potential of different ratios of lipids and dexamethasone were determined by dynamic light scattering (DLS) using a Malvern Zeta Sizer Nano series .2. For the macrophage polarization, M1 macrophages were obtained by lipopolysaccharide  treatment for 12 h  containing 10% (v/v) fetal bovine serum , 100 Units/mL penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere of 95% air with 5% COfor 12 h  and M2 mfor 12 h .4 cells/mL per well for 24 h before treatment and then incubated for another 24 h in the presence of different concentrations of Dex-L. The cell viability was measured using the MTT . After discarding the supernatant, each well was added 10% MTT and incubated for 4 h in the dark. The MTT solution was removed and then the formazan solution was added and shaking for 10 min. The absorbance was measured at 490 nm with a Microplate reader .The RAW264.7 cells were seeded into 96-well plates at a density of 3 × 10Dexamethasone was labeled with FITC (Fluorescein Isothiocyanate), a fluorescein and the drug loading of macrophages could be determined by the fluorescence intensity of FITC-labeled dexamethasone. Macrophages were incubated with FITC fluorescence-labeled dexamethasone liposomes and Triton X-100 was used to disrupt the cell supernatants at 0, 2, 4, 6, and 8 h. Following the fluorescence intensity of cell lysis solutions with different times were detected at excitation wavelength of 490 nm and emission wavelength of 515 nm. After macrophages were incubated with FITC fluorescence-labeled dexamethasone liposomes for 4 h, the liquid from the wells were removed. The phenol-free red blood cell culture medium containing 1% serum was added and the fluorescence intensity of supernatants at 0, 2, 4, 6, 8, and 10 h were tested under the same fluorescence detection conditions.In our experiment, we used 24 mm Transwell® with 8 μm Pore Polyester Membrane Insert from Corning Company. The 3T3 cells were implanted in a 24-well plate. Before co-culture, macrophages and dexamethasone liposomes were allowed to interact 4 h in advance to complete the dexamethasone liposome payload. Then, the chamber was put into a 24-well co-culture plate and TGF-β1 (5 ng/mL) was added to the medium to induce the migration and activation of fibroblasts . AnotherDraw three straight lines after the 24-well plate, and insert the cells into the well plate at a suitable density. After the cells adhere to the wall, use a 200 μL pipette to make a scratch perpendicular to the three straight lines. Images were taken with an inverted microscope  to record cell migration at 0, 24, and 48 h.For immunofluorescence staining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, the cells were treated with 2% BSA at 37°C for 1.5 h and incubated with the indicated antibodies at 4°C overnight. After being washed 3 times in PBS, the cells were stained with the corresponding secondary antibodies Alexa Fluor 488-conjugated rabbit anti-mouse antibody. Nuclei were stained with Hoechst 33342. Images were obtained with Operetta High Content Analysis (HCA) System . Quantitative analysis of fluorescence by ImageJ.Macrophages were incubated with dexamethasone liposomes for 4 h and the concentration of dexamethasone was 100 μM. The macrophages were gently scraped off with a cell scraper and resuspended in PBS. Then the cells were injected into mice via tail vein.Idiopathic pulmonary fibrosis was established in mice model by intratracheal instillation of bleomycin (BLM)-A5 hydrochloride  (1.5 mg/kg dissolved in PBS). Mice were frequently examined by micro-CT after the treatment of BLM for 5th days. Mice presenting fibrosis lesions involving more than 25% of the lung were further divided into five experiment groups (6 mice per group), treating once daily with 3 mg/kg Dex . All the mice were administered via intravenous injection. Micro-CT scans were performed at baseline, then to evaluate the effect of these treatments. Lung tissues were quartered and processed for the following experiments: the left lobe was inflated and fixed in 10% buffered formalin for histological and immunohistochemical examination, and the remaining lobes were stored at −80°C and used for preparation of whole lung tissue protein extracts.The cell supernatant and mouse serum were centrifuged at 3,000 rpm for 10 min to be collected. The levels of TGF-β1 and IL-6 in the mice serum and cell-free supernatants were measured using the ELISA kits  according to the manufacturer's instructions and the cytokine concentrations were calculated using standard curves.Mice lung tissues (50 mg) were homogenized, centrifuged and the supernatant was collected. Blood was collected from eyeball venous plexus of mice, and the serum stored at 4°C. IL-6, TGF-β1, IL-1β, and Arg-1 were detected by ELISA kits  according to the manufacturer's instructions.The whole blood was obtained from the eye orbit of mice and stored in an anticoagulant tube containing EDTA. Neutrophils and lymphocytes were detected using a blood analyzer .Mice were anesthetized by Tribromoethanol; a small incision was made on the trachea and BAL fluid was collected by cannula. Briefly, cannula was inserted into trachea with sufficient ice-cold PBS (0.5 mL each time) to collect BALF. The procedure was repeated thrice, and 70–80% recovery of collected BALF was observed. After collection of BALF, Later, BAL fluid was centrifuged at 4,000 rpm for 10 min at 4°C, and supernatant was used for determination of IL-6, TGF-β1, and IL-1β levels in BALF were detected by ELISA.2O2 for 10 min, followed by incubation with rabbit anti-CD206 at 4°C overnight. In addition, the slides were stained with H&E for structured observation, or with Masson's trichrome stain for detection of collagen deposits according to the instructions by the manufacturer.Immunohistochemistry was performed on 4 μm, paraffin embedded lung tissue and mounted on polylysine-coated slides. The slides were cleared of paraffin and subjected to antigen retrieval . Next, quenching of endogenous peroxidase activity was achieved by incubation with 3% (v/v) HThe measurement of hydroxyproline was conducted with a hydroxyproline measurement kit (NanJing JianCheng Bioengineering Institute) according to the manufacturer's instructions. Approximately 30 mg (wet weight) lung tissue was collected. One milliliter of alkaline hydrolysate was added and the tissue was boiled at 95°C for 20 min with constant mixing. The pH was adjusted to 6.0–6.8 using the reagent provided. About 3–4 mL of supernatant was collected for measurement after sorption onto active carbon. The hydrolysate was centrifuged at 3,500 rpm for 10 min. One milliliter of supernatant was then carefully taken for measurement according to the manufacturer's instructions.post-hoc Tukey's-test. The statistical significance for all tests was set at P-values of <0.05.All analyses were performed using SPSS 7.0 software  or GraphPad . The results were expressed as the mean ± standard deviation. Multi-group comparisons of the means were carried out by one-way analysis of variance (ANOVA) test with in situ  was swallowed and prepared by the thin film dispersion method, the particle size of Dex-L measured by dynamic light scattering is 125.27 ± 1.07 nm , and TheNext, in order to measure the drug loading efficacy, RAW264.7 cells were treated by the FITC fluorescence-labeled dexamethasone (f-Dex) and Rh-PE fluorescence-labeled liposomes for different times. After measuring the fluorescence intensity of FITC and Rh-PE by Operetta High Content Analysis (HCA) System, we found that macrophages were incubated with f-Dex-L for 4 h leading to the highest effective intracellular uptake of Dex, while prolonged incubation time only slightly increased uptake of Dex .The macrophages were incubated with liposomes containing f-Dex at different concentrations and the drug uptake of macrophages was concentration dependent . The uptPolarized macrophages (M1 and M2 macrophages phenotypes) play important roles during the development of fibrosis. To investigate the effects of Dex-L on the polarization status of macrophages, Dex-L were incubated with M1 and M2 macrophage and subsequently cytokine cues such as nitric oxide synthase (iNOS), IL-6 and IL-10 were determined.p < 0.001) and 15%  respectively, indicating the M1 polarization was inhibited. p < 0.01) and 22.6%  respectively. p < 0.001). The liposomes without drug at a concentration of 100 μM sharply reduced the expression of CD206 in macrophages, indicating the inhibition effect during the process of M2 macrophage polarization (As shown in rization . All thep < 0.001) than the group treated with TGF-β1 inhibitor SB431542 . Similarly, the migration assay showed that Dex-L-MVs were capable of significant inhibition rate comparing to activated fibroblast cells  using hematoxylin and eosin (H&E) staining. The results showed that no pathological changes in organs after 10 days treatment, demonstrating that systemic toxicity was rarely exhibited by intravenous injection of the Dex-L-MVs groups  . We detes groups . Next, ws groups . Moreoves groups . Togethep < 0.001) (p < 0.001), after treatment with Dex-L-MVs, the expression of Arg-1 was significantly decreased , which further presented the immune-modulatory effect of our macrophage delivery system containing DEX. We also evaluated the content of hydroxyproline (HYP) in the lung tissues, and found that Dex-L-MVs sharply reduced the amount of collagen in the lungs of BLM treated mice  is essentially diagnostic of IPF in the appropriate clinical setting. Recently micro-CT (μCT) has been used to quantify the pulmonary fibrosis in mice, and the image quality of the μCT of each mouse was assessed semi-quantitatively on a four-point ranking scale as previously described , 25. The< 0.001) . In addi< 0.001) . The expted mice .in vivo.During the development of fibrosis, the inflammatory response will drive the process of fibrosis . H&E stain vivo by the suppression of inflammatory cytokines  and pirfenidone (PFD) were clinically approved. Because of their strong side effect, there is an urgent need to develop safer therapeutic approaches to suppress the progression of IPF. So far, only few designed targeted drug delivery systems using particles for IPF are reported, all attempts in targeted drug delivery have relayed on the specific recognition to the surface receptor. For example, Chang employed matrix metalloproteinase-2 (MMP-2) responsive peptide (peptide E5)-modified engineered liposomes loaded with nintedanib (NIN) and colchicine (COL) that can firstly target endogenous monocyte-derived multipotent cells (MOMCs) and then be selectively delivered into IPF lungs . Liposomytokines . These dytokines , 29.Macrophages or monocytes can respond to chemotactic cues and migrate to inflammatory sites, making them a potentially attractive drug delivery vehicle. The macrophages have used their powerful phagocytic function to deliver drugs to the hypoxic region of tumors in the form of “Trojan horses” –32. Duriin vitro and in vivo. With the help of macrophage membranes or membrane-like membranes to specifically intervene in the immune response via the specific binding on the cell membrane (in vitro experiments. Similar to M2 macrophage polarization, interestingly, Dex-L was observed to exhibit an inhibitory activity on the polarization of M1 macrophages by decreased iNOS and IL-6 levels in vitro. We anticipate that this cell-based drug delivery strategy will speak new in vitro, in vivo in the field of endogenous immune targeting IPF therapy.Here, we developed a macrophage-based delivery system loading dexamethasone liposomes to attenuate pulmonary fibrosis membrane , 36, we The original contributions generated in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the Tianjin International Joint Academy of Biotechnology and Medicine.XS performed experiments, analyzed data, and wrote the manuscript. YW analyzed the data. JY designed of the study, reviewed data, and contributed to the project conception and manuscript revision. YZ, GF, and FT provided guidance and partial funding support. ZX revised the manuscript. All authors reviewed, revised, and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.